Methods for inhibiting conversion of choline to trimethylamine (tma)

ABSTRACT

A method of inhibiting the conversion of choline to trimethylamine (TMA) and lowering TMAO in an individual by providing a composition comprising a compound set forth in Formula (I):

FIELD OF THE INVENTION

The invention generally relates to materials and methods for inhibitingtrimethylamine production in an individual.

BACKGROUND

Trimethylamine (TMA) and its derivative trimethylamine N-oxide (TMAO)are metabolites linked to disorders such as kidney disease, diabetesmellitus, obesity, trimethylaminuria, and cardiovascular disease (CVD).TMA is produced in the gut by bacteria which are capable of convertingsubstrates including but not limited to choline, to TMA. There is anunmet need for compounds which inhibit the production of TMA bybacteria.

CVD is a general term encompassing a range of conditions affecting theheart and blood vessels, including atherosclerosis, coronary heartdisease, cerebrovascular disease, heart failure, cardiomyopathy,atherothrombotic disease, aorto-iliac disease, and peripheral vasculardisease. CVD is generally associated with conditions that involvenarrowed, blocked, aneurysmal or dissection of one or more bloodvessels, or thrombosis (blood clot formation). Complications associatedwith CVD include, but are not limited to, myocardial infarction, stroke,angina pectoris, acute coronary syndrome, transient ischemic attacks,congestive heart failure, aortic aneurysm, atrial fibrillation orflutter, ventricular arrhythmias, cardiac conduction abnormalities, needfor revascularization and death. Revascularization can include but isnot limited to angioplasty, stenting, coronary artery bypass grafting,repair or replacement of vascular shunt or access such as anarteriovenous fistula. Complications associated with atherothromboticdisease include, but are not limited to, myocardial infarction, stroke,pulmonary embolism, deep venous thrombosis. According to the WorldHealth Organization, CVDs are the leading cause of death globally, withover 75% of deaths occurring in low- and middle-income countries. WorldHealth Organization Fact Sheet No. 317, updated January 2015. The WorldHealth Organization projects that diabetes will be the seventh leadingcause of death in 2030. World Health Organization Fact Sheet No. 312,updated January 2015. Prevention and management of conditions associatedwith TMA and TMAO, including CVD and diabetes, is a major public healthconcern.

SUMMARY OF THE INVENTION

The disclosure is based, at least in part, on the discovery thatcompounds of Formula (I) inhibit choline metabolism by gut microbiotaresulting in reduction in the formation of trimethylamine (TMA). Thedisclosure provides compositions and methods for, e.g., inhibiting theconversion of choline to TMA in vitro and in vivo, for improving ormaintaining cardiovascular, cerebrovascular, or peripherovascularhealth, and for improving or preventing a condition associated with TMAand TMAO. In certain aspects, the invention provides one or more methodsof inhibiting the conversion of choline to TMA in an individual.

In certain aspects, the invention provides one or more methods ofreducing the production of TMAO comprising inhibiting the conversion ofcholine to TMA by a bacterium, by providing one or more compounds as setforth in Formula (I). The invention provides a method of inhibiting theconversion of choline to TMA in an individual. The method comprisesadministering to the individual a composition comprising a compound setforth in Formula (I):

wherein:

W is selected from F, Cl, Br or I; X⁻ is a pharmaceutically acceptableanion, and salts or solvates thereof.

The compound of Formula (I) can be administered in an amount effectiveto inhibit conversion of choline to TMA and TMAO in the individual.

The invention further provides a method of improving or maintainingcardiovascular health. A method may comprise administering to theindividual a composition comprising a compound as set forth in Formula(I), as described herein in an amount that improves or maintainscardiovascular health. The invention also provides a method of improvinga condition associated with the conversion of choline to trimethylamine(TMA) in an individual. The method comprises administering to theindividual a composition comprising a compound as set forth in Formula(I), as described herein in an amount effective to improve thecondition. In some embodiments, the condition may be trimethylaminuria,reduced or impaired kidney function, kidney disease, chronic kidneydisease (CKD), end-stage renal disease (ESRD), diabetes mellitus,obesity, or cardiovascular disease, such as angina, arrhythmia,atherosclerosis, cardiomyopathy, congestive heart failure, coronaryartery disease (CAD), carotid artery disease, endocarditis, coronarythrombosis, myocardial infarction (MI), high bloodpressure/hypertension, hypercholesterolemia/hyperlipidemia, peripheralartery disease (PAD), or stroke. In some embodiments, the condition isadverse ventricular remodeling, ventricular systolic dysfunction,ventricular diastolic dysfunction, cardiac dysfunction, ventriculararrhythmia, or cardiovascular disease or atherosclerosis due to oralbiofilm formation and periodontal disease.

The invention further provides the compounds of Formula (I) for use ininhibiting the conversion of choline to TMA in vivo or in vitro, forimproving or maintaining cardiovascular health, and for improving acondition associated with the conversion of choline to TMA; and use ofthe compounds of Formula (I) for inhibiting the conversion of choline toTMA in vivo or in vitro, for improving or maintaining cardiovascularhealth, and for improving a condition associated with the conversion ofcholine to TMA.

The foregoing summary is not intended to define every aspect of theinvention, and additional aspects are described in other sections, suchas the Detailed Description. In addition, the invention includes, as anadditional aspect, all embodiments of the invention narrower in scope inany way than the variations defined by specific paragraphs set forthherein. For example, certain aspects of the invention that are describedas a genus, and it should be understood that every member of a genus is,individually, an aspect of the invention. Also, aspects described as agenus or selecting a member of a genus should be understood to embracecombinations of two or more members of the genus. In certain aspects,the invention may be described as related to a substrate, for examplecholine, and may also relate to metabolites or precursors of saidsubstrate, for example precursors or metabolites of choline such aslecithin or glycerophosphocholine. With respect to aspects of theinvention described or claimed with “a” or “an,” it should be understoodthat these terms mean “one or more” unless context unambiguouslyrequires a more restricted meaning. The term “or” should be understoodto encompass items in the alternative or together, unless contextunambiguously requires otherwise, for example X or Y, means X or Y orboth. If aspects of the invention are described as “comprising” afeature, embodiments also are contemplated “consisting of” or“consisting essentially of” the feature.

DETAILED DESCRIPTION OF THE INVENTION

The components of the present compositions are described in thefollowing paragraphs.

The present invention provides one or more methods of reducing theproduction of TMA comprising: inhibiting the conversion of choline toTMA by a bacterium using a composition comprising a compound set forthin Formula (I). The present invention also provides synthesis methods toproduce a series of selected haloalkyl amine derivatives, as exemplifiedin Formula (I). Such compounds may be used to inhibit the conversion ofcholine to TMA in vivo or in vitro, or inhibit the production of TMA bybacteria. The compounds of Formula (I) may be administered to anindividual in an amount effective to inhibit the production of TMA andTMAO by bacteria in the gut of an individual, for example fromsubstrates including but not limited to choline.

TMA synthesized by bacteria resident in the gut of mammals is oxidizedin the liver to trimethylamine N-oxide (TMAO, TMANO). Exemplaryprecursors of TMA include choline, betaine, phosphatidylcholine,phosphocholine, glycerophosphocholine, carnitine, L-carnitine, TMAO,sphingomyelin, and lecithin, many of which are derived from dietarysources such as, for example, whole eggs and beef liver. These sourcesmay act as substrates for bacteria that can metabolize them to TMA.Without wishing to be bound to a particular mechanism or biochemicalpathway, the anaerobic conversion of choline to TMA is facilitated by aglycyl radical enzyme homologue, choline trimethylamine-lyase (CutC).Craciun et al., Proc. Natl. Acad. Sci. (2012), 109: 21307-21312. Thereduction of choline conversion to TMA by bacteria in the gut of anindividual leads to a reduction in TMA absorption from the gut, leadingto a subsequent reduction in plasma TMAO following oxidation of TMA toTMAO by the flavin monooxygenase 3 (FMO3) enzyme in the liver. Wang etal., Nature (2011), 472: 57-63. Lower plasma TMAO levels are related toa lower incidence of major cardiovascular events in humans. Tang et al.,NEJM (2013) 368: 1575-1584. The conversion of choline to TMA may bemediated by one species of bacteria or comprise a multi-step processinvolving two, three or more species of bacteria.

As described previously, the present invention is based, at least inpart, on the discovery that compounds of Formula (I) interfere withcholine metabolism by gut microbiota resulting in reduction in theformation of TMA and trimethylamine N-oxide (TMAO). The disclosureprovides compositions and methods that for example inhibit theconversion of choline to TMA in vitro and in vivo, improve or maintaincardiovascular, cerebrovascular, and peripherovascular health, andimprove or prevent a condition associated with increased TMA and TMAO.Other conditions associated with increased levels of TMA may includeproduction of TMA by bacteria in the vagina leading to vaginal odor, orproduction of TMA by bacteria on the body leading to body odor, orproduction of TMA by bacteria in the mouth leading to bad breath or oralcare biofilm development, or during pregnancy where the third trimesterand post-partum period are associated with an increased risk ofthrombosis, thus lowering TMA and TMAO levels may reduce this risk. Thedisclosure additionally provides compositions and methods to increasethe availability of choline in the gut of an individual with a conditionwhere increased choline availability would be beneficial, by inhibitingcholine catabolism. One such condition is during pregnancy and thepost-partum period where increased choline availability in the gut ofthe mother may promote brain development for the fetus and newborn.

Conversion of choline to TMA by gut bacteria has been attributed to theglycyl radical enzyme homologue, choline trimethylamine-lyase CutC.Craciun et al. (2014) ACS Chem Biol 9: 1408-1413. It has been describedthat not all gut microbes contain the gene cluster including CutC.Martinez-del Campo et al. (2015) mBio 6(2):e00042-15.doi:10.1128/mBio.00042-15. The cut gene cluster contains a set of genesencoding the glycyl radicle enzyme CutC, and a glycyl radicle activatingprotein CutD, cutC/D gene cluster. Craciun et al. (2012) PNAS109:21307-21312.

In contrast, most sequenced bacteria convert choline to glycine betaine(GB, or trimethylglycine) which primarily acts as an osmoprotectant.Additionally, some bacteria can convert choline to GB and then toglycine, which may be used as a source of carbon and nitrogen. Wargo(2013) Appl. Environ. Microbiol. 79:2112-2120. Pseudomonas aeruginosa isone such species of bacteria that can convert choline to glycine via GB,dimethyl glycine (DMG) and sarcosine.

All percentages and ratios used hereinafter are by weight of totalcomposition, unless otherwise indicated. All percentages, ratios, andlevels of ingredients referred to herein are based on the actual amountof the ingredient, and do not include solvents, fillers, or othermaterials with which the ingredient may be combined as a commerciallyavailable product, unless otherwise indicated.

All measurements referred to herein are made at 25° C. unless otherwisespecified.

The components of the present compositions are described in thefollowing paragraphs.

As used herein, “dose” refers to a volume of medication, such as liquidmedication or oral dosage unit, containing an amount of a drug activesuitable for administration on a single occasion, according to soundmedical practice. A dose can be orally administered. In one example, adose can be a liquid medication and can be about 30 mL, in anotherexample about 25 mL, in another example about 20 mL, in another exampleabout 15 mL, and in another example about 10 mL, and in another exampleabout 5 mL. In another example, a dose of liquid medication can be fromabout 5 mL to about 75 mL, in another example from about 10 mL to about60 mL, in another example from about 15 mL to about 50 mL, in anotherexample from about 25 mL to about 40 mL, and in another example fromabout 28 mL to about 35 mL. In another example, the dose can be a soliddosage form and can be from about 25 mg to about 5 g, in another examplefrom about 100 mg to about 3 g, in another example from about 250 mg toabout 2 g, in another example from about 500 mg to about 1.6 g, and inanother example from about 750 mg to about 1 g. In addition, a dose maybe a solid dosage form wherein one dose is about 3 g or a dose can beabout 1.6 g. The concentration of active ingredients can be adjusted toprovide the proper doses of actives given the liquid or solid dose size.In certain embodiments, a dose can be administered about every 4 hours,about every 6 hours, about every 8 hours, about every 12 hours, or aboutevery 24 hours.

As used herein, “medication” refers to compositions comprising acompound of Formula (I), such as pharmaceuticals, including prescriptionmedications, over-the-counter medications, behind-the-countermedications and combinations thereof. In some examples, a medication canbe a dietary supplement which can contain vitamins, minerals, andsupplements (VMS) including supplements or ingredients such asbotanicals.

Medication compositions can be in any suitable form including liquidcompositions and solid oral dosage forms. Non limiting examples ofliquid compositions can include syrups, beverages, supplemental water,foam compositions, gel compositions, particles suspended in a liquidformulation, a solid in a gelatin or foam, saline wash and combinationsthereof. Non-limiting examples of solid oral dosage forms can includetablets, capsules, caplets, sachets, sublingual dosage forms, buccaldosage forms, soft gels, and other liquid filled capsules, dissolvabledosage forms including dissolvable strips, films, gums including acenter filled gum, gummies including a center filled gummy, lozenges,center filled tablets, powder, granules, pellets, microspheres,nanospheres, beads, or nonpareils, and combinations thereof. Tablets caninclude compressed tablets, chewable tablets, dissolvable tablets, andthe like. In some examples, the medication can be applied to the skin,in an ointment such as a petroleum jelly based ointment. In someexamples the medication may be provided in a delivery device. In otherexamples, the medication can be inhaled, such as a nose spray orinhaler. In still other examples, the medication can be in a drink, suchas a warm beverage. In further examples, the medication can contain apharmaceutical active.

The medications can be in a form that is directly deliverable to themouth, throat, or skin. In some embodiments, the medication compositionscan be delivered by a delivery device selected from droppers, pump,sprayers, liquid dropper, saline wash delivered via nasal passageway,cup, bottle, canister, pressurized sprayers, atomizers, air inhalationdevices, squeezable sachets, power shots, blister cards, and otherpackaging and equipment, and combinations thereof. The sprayer,atomizer, and air inhalation devices can be associated with a battery orelectric power source.

As used herein the term “individual” includes both humans and othertypes of mammals sharing the TMAO pathway, such as domesticated animals,including but not limited to, domestic dogs (canines), cats (feline),horses, cows, ferrets, rabbits, pigs, rats, mice, gerbils, hamsters,horses, and the like.

A wide variety of individuals may wish to reduce the level of TMAproduced by bacteria in their digestive tract. For example, individualsdiagnosed with cardiovascular disease may be directed by a physician totake prescription drugs or effect lifestyle changes to modulate bloodcholesterol levels to reduce the risk of serious cardiovascular events.Other individuals not previously diagnosed with cardiovascular diseasebut who wish to improve or maintain cardiovascular health may also wishto reduce the level of TMA produced by digestive tract bacteria. Asdescribed further herein, a reduction in TMA (and, by extension, TMAO)is achieved by the compositions described herein, which may include, forexample, a dietary supplement comprising the compounds of Formula (I).

The disclosure includes, a method of inhibiting the conversion ofcholine to TMA, a method of improving cardiovascular health, and amethod of improving a condition associated with conversion of choline toTMA comprising administering to the individual a composition comprisinga compound of Formula (I). Features of the compositions and methods aredescribed below. Section headings are for convenience of reading and notintended to be limiting per se. The entire document is intended to berelated as a unified disclosure, and it should be understood that allcombinations of features described herein are contemplated, even if thecombination of features are not found together in the same sentence, orparagraph, or section of this document. It will be understood that anyfeature of the methods or compounds described herein can be deleted,combined with, or substituted for, in whole or part, any other featuredescribed herein.

Compounds

The methods of the present invention may comprise administering to theindividual a composition comprising a compound set forth in Formula (I):

wherein:

W is selected from F, Cl, Br or I; X⁻ is a pharmaceutically acceptableanion, and salts or solvates thereof.

In certain embodiments, the compound (as shown in TABLE 2 herein) may beselected from the group consisting ofN-(chloromethyl)-2-fluoro-N,N-dimethylethan-1-aminium chloride,2-fluoro-N-(iodomethyl)-N,N-dimethylethan-1-aminium chloride,(2-fluoroethyl)(fluoromethyl)dimethyl-14-azane chloride andN-(bromomethyl)-2-fluoro-N,N-dimethylethan-1-aminium chloride, and saltsor solvates thereof.

In certain embodiments, the compound may be selected from the groupconsisting of N-(chloromethyl)-2-fluoro-N,N-dimethylethan-1-aminium,2-fluoro-N-(iodomethyl)-N,N-dimethylethan-1-aminium,(2-fluoroethyl)(fluoromethyl)dimethyl-14-azane,N-(bromomethyl)-2-fluoro-N,N-dimethylethan-1-aminium, and apharmaceutically acceptable anion and salts and solvates thereof.

The compound is administered in an amount effective to achieve thedesired effect, e.g., inhibit conversion of choline to TMA, improve ormaintain cardiovascular health, or improve a condition associated withconversion of choline to TMA.

The invention further provides for methods to synthesize selectedhaloalkyl amine derivatives as representatives of Formula (I). Suchcompound derivatives may also be used to inhibit the production of TMAby a bacterium or for inhibiting the conversion of choline to TMA invivo or in vitro, by providing a composition comprising a composition asset forth in Formula (I).

Compounds of Formula (I) can be synthesized using the general Scheme 1,shown below.

wherein

X⁻ is a pharmaceutically acceptable anion and n is 1, 2 or 3 andincluding any acceptable salts or solvates thereof;comprising the steps of reacting Compound A:

with a compound of structure B:

wherein LG is any suitable leaving group known to one skilled in theart,to form a compound of Formula (I); and any acceptable salts or solvatesthereof.X⁻ may be an anion capable of forming a salt with an ammonium group. Incertain embodiments, X⁻ is a pharmaceutically acceptable anion selectedfrom chloride, bromide, iodide, phosphate, and sulfate salts. Additionalpharmaceutically acceptable acid addition salts include, for example,succinate, maleate, tartrate, citrate, glycolate, andtrifluoromethanesulfonate or triflate, thus X⁻ may be selected fromsuccinate, maleate, tartrate, citrate and glycolate. X⁻ is preferably achloride, bromide, iodide, trifluoromethanesulfonate or triflate, saltform. When the compound of interest exists as a sulfoxide, then X⁻ isabsent. When the compound of interest exists as a carboxylate, then X⁻is absent.

“Alkyl” refers to straight chained and branched saturated hydrocarbongroups containing 1-30 carbon atoms (i.e., C₁-C₃₀), for example, 1-20carbon atoms (i.e., C₁-C₂₀) or 1-10 carbon atoms (i.e., C₁-C₁₀). Invarious embodiments, the alkyl groups of Formula (I) are independentlyselected from C₁-C₄ alkyls, i.e., alkyl groups having a number of carbonatoms encompassing the entire range (i.e., 1 to about 4 carbon atoms),as well as all subgroups (e.g., 1-2, 1-3, 1-4, 2-3, 2-4, 3-4, 1, 2, 3,and 4 carbon atoms). Nonlimiting examples of alkyl groups include allyl,methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl),t-butyl (1,1-dimethylethyl) and propargyl. Unless otherwise indicated,an alkyl group can be an unsubstituted alkyl group or a substitutedalkyl group. Alkyl groups may also be substituted, for example, with oneor more of hydroxy (OH), alkoxy, carboxy, cycloalkyl, heterocycloalkyl,and halo.

The term “heteroalkyl” is defined the same as alkyl except thehydrocarbon chain or branched chain contains one to three heteroatomsindependently selected from oxygen, nitrogen or sulfur. The terms“heterocycloalkyl” or “heterocyclic” are defined similarly ascycloalkyl, except the ring contains one to three heteroatomsindependently selected from oxygen, nitrogen, or sulfur. Nonlimitingexamples of heterocycloalkyl groups include piperdine, tetrahydrofuran,tetrahydropyran, 4H-pyran, dihydrofuran, morpholine, thiophene,1,4-dioxane, furan, pyridine, pyrrole, pyrrolidine, imidazole, pyrazole,triazole, thiazole, pyrazine, pyran, oxazole, oxazine, thiazine,pyrimidine, and the like. Cycloalkyl and heterocycloalkyl groups can besaturated or partially unsaturated ring systems optionally substitutedwith, for example, one to three groups, independently selected alkyl,alkenyl, OH, C(O)NH₂, NH₂, oxo (═O), aryl, haloalkyl, halo, and alkoxy.Heterocycloalkyl groups may also be further N-substituted with alkyl,hydroxyalkyl, alkoxyaryl, alkylenearyl, and alkyleneheteroaryl.

The terms “cycloalkyl” or “carbocyclic” refer to an aliphatic cyclichydrocarbon group containing 3-8 carbon atoms (e.g., 3-5, 5-8, 3, 4, 5,6, 7, or 8 carbon atoms). Nonlimiting examples of cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,and cyclooctyl. Unless otherwise indicated, a cycloalkyl group can be anunsubstituted cycloalkyl group or a substituted cycloalkyl group.

The term “hydroxy” or “hydroxyl” refers to a “—OH” group. The term“amino” or “amine” refers to a —NH₂, or a —NH— group. “Amine” includescyclic amines optionally substituted with one or more additionalheteroatoms. The term “carboxy” or “carboxyl” refers to a “—COOH” group.The term “thiol” or “sulfhydryl” refers to a “—SH” group. The term“cyano” refers to a —C≡N group, also designated —CN.

A “substituted” alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl,heteroaryl, or alkoxyl, refers to an alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, or alkoxyl having at leastone hydrogen radical that is substituted with a non-hydrogen radical(i.e., a substituent). Examples of non-hydrogen radicals (orsubstituents) include, but are not limited to, alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, ether, aryl, heteroaryl,heterocycloalkyl, hydroxyl, oxy (or oxo), alkoxyl, ester, thioester,acyl, carboxyl, cyano, nitro, amino, amido, or sulfur. When asubstituted alkyl group includes more than one non-hydrogen radical, thesubstituents can be bound to the same carbon or two or more differentcarbon atoms.

Physiologically acceptable salts of haloalkyl amine derivatives arecontemplated and can be formed by reacting an ammonium compound with analkylating agent containing a leaving group. Leaving groups commonlyemployed in alkylation reactions with sulfur are known in the art.Leaving groups such as, but not limited to those skilled in the art,include the halides (chlorine, bromine, iodine, etc.) and sulfonateesters of alcohols (tosylate, mesylate, cumenesulfonate, triflate,etc.). Physiologically accepted salts can be formed directly from thealkylation reaction of sulfur with an alkylating agent or can beprepared by an ion exchange process. Physiologically accepted saltsinclude but are not limited to haloalkyl amine halides, phosphates,carboxylates, and sulfonates.

Salts, such as physiologically acceptable salts, of the disclosedcompounds are contemplated and may be prepared by reacting theappropriate base or acid with a stoichiometric equivalent of thecompound. Acids commonly employed to form physiologically acceptablesalts include inorganic acids such as hydrogen bisulfide, hydrochloricacid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoricacid, as well as organic acids such as para-toluenesulfonic acid,salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleicacid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formicacid, glutamic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, cumenesulfonic acid, lactic acid, oxalic acid,para-bromophenylsulfonic acid, carbonic acid, succinic acid, citricacid, benzoic acid and acetic acid, as well as related inorganic andorganic acids. Physiologically acceptable salts include sulfate,pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, trifluoromethanesulfonate ortriflate, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,terephthalate, sulfonate, xylene sulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate, O-hydroxybutyrate,glycolate, maleate, tartrate, bitartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate and other salts. Physiologically acceptable acid additionsalts include, for example, those formed with mineral acids such ashydrochloric acid and hydrobromic acid and those formed with organicacids such as maleic acid.

Physiologically acceptable base addition salts may be formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Physiologically acceptable salts of compounds may also be prepared witha physiologically acceptable cation. Physiologically acceptable cationsthat can be used are well known in the art and include alkaline,alkaline earth, ammonium and quaternary ammonium cations. Carbonates orhydrogen carbonates are also options in this regard. Examples of metalsused as cations are sodium, potassium, magnesium, ammonium, calcium,ferric, and the like. Examples of amines that can be used include, butare not limited to, isopropylamine, histidine,N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.

In a further embodiment, the compound is a stable isotope variant, forexample wherein deuterium is substituted for one or more of thehydrogens.

In various embodiments, the compound of Formula (I) demonstrates an IC₅₀of 1×10⁻³ or less, 5×10⁻³ or less, 1×10⁻⁴ or less, 5×10⁻⁴ or less,1×10⁻⁵ or less, 5×10⁻⁵ or less, or 1×10⁻⁶ or less, or 1×10⁻⁷ or less, or1×10⁻⁸ or less, or 1×10⁻⁹ or less, or 1×10⁻¹⁰ or less or 1×10⁻¹¹ or lessor 1×10⁻¹² or less, or between 1×10⁻⁹ and 1×10⁻³, or between 1×10⁻¹² and1×10⁻⁹, or between 1×10⁻⁹ and 1×10⁻⁶, or between 1×10⁻⁸ and 1×10⁻⁶, orbetween 1×10⁻⁶ and 1×10⁻³, between 1×10⁻⁶ and 1×10⁻⁴, between 1×10⁻⁶ and1×10⁻⁵, between 1×10⁻⁵ and 1×10⁻³, or between 1×10⁻⁴ and 1×10⁻³, orbetween 1.7×10⁻¹¹ and 1×10⁻⁷, (observed 50% inhibition of TMA (or TMAO)formation from choline; mol/L), in the assays described in EXAMPLE 2 orEXAMPLE 5. In various embodiments, the compound of Formula (I)demonstrates an IC₅₀ of between 1×10⁻¹¹ and 1×10⁻⁷, or between 1×10⁻⁸ to1×10⁻³, or between 1.2×10⁻⁶ to 2×10⁻³, or between 1×10⁻⁶ to 1×10⁻⁴(observed 50% inhibition of TMA formation from choline; mol/L) asmeasured in the assays described in EXAMPLE 2 or EXAMPLE 5.

In various embodiments, the compound of Formula (I) demonstrates an EC₅₀of 1×10⁻³ or less, 5×10⁻³ or less, 1×10⁻⁴ or less, 5×10⁻⁴ or less,1×10⁻⁵ or less, 5×10⁻⁵ or less, or 1×10⁻⁶ or less, or 1×10⁻⁷ or less, or1×10⁻⁸ or less, or 1×10⁻⁹ or less, or 1×10⁻¹⁰ or less or 1×10⁻¹¹ or lessor 1×10⁻¹² or less, or between 1×10⁻⁹ and 1×10⁻³, or between 1×10⁻¹² and1×10⁻⁹, or between 1×10⁻⁹ and 1×10⁻⁶, or between 1×10⁻⁸ and 1×10⁻⁶, orbetween 1×10⁻⁶ and 1×10⁻³, between 1×10⁻⁶ and 1×10⁻⁴, between 1×10⁻⁶ and1×10⁻⁵, between 1×10⁻⁵ and 1×10⁻³, or between 1×10⁻⁴ and 1×10⁻³, orbetween 1.7×10⁻¹¹ and 1×10⁻⁷, (observed 50% inhibition of TMA (or TMAO)formation from choline; mg/kg), in the assays described in EXAMPLE 6. Invarious embodiments, the compound of Formula (I) demonstrates an IC₅₀ ofbetween 1×10⁻¹¹ and 1×10⁻⁷, or between 1×10⁻⁸ to 1×10⁻³, or between1.2×10⁻⁶ to 2×10⁻³, or between 1×10⁻⁶ to 1×10⁻⁴ (observed 50% inhibitionof TMA formation from choline; mg/kg) as measured in the assaysdescribed in EXAMPLE 6.

The invention includes a method of inhibiting the conversion of cholineto TMA in an individual which may comprise administering to anindividual a composition comprising a compound set forth in Formula (I),as described previously. In certain embodiments, as described herein, anindividual may be in need of reduced TMA levels, improvement ofcardiovascular health, and the like. An individual may exhibit anelevated level of TMA or a metabolite thereof (e.g., TMAO, dimethylamine(DMA), or monomethylamine (MMA)) prior to administration. In variousembodiments, an individual suffers from cardiovascular disease, ingestsa diet high in choline, or exhibits one or more CVD risk factors (e.g.,smoking, stress, high total cholesterol, high LDL cholesterol, low HDLcholesterol, age, hypertension, family history of CVD, obesity,prediabetes, diabetes, or the like).

A method of inhibiting the conversion of choline to TMA in vitro is alsocontemplated. For example, a method may comprise contacting a bacterium,such as a bacterium that is represented in the gut microflora, or abacterial lysate that metabolizes choline to produce TMA with a compoundof Formula (I), as described previously. In various embodiments, abacterium may be selected from Proteus mirabilis, Desulfovibrioalaskensis, Clostridium ljungdahlii, C. scindens, C. aldenense, C.aminobutyricum, Collinsella tanakaei, Anaerococcus vaginalis,Streptococcus dysgalactiae, Desultitobacterium hafniense, Klebsiellavariicola, K. pneumonia, P. penneri, Eggerthella lenta, Edwardsiellatarda, Escherichia coli, E. fergussonii, or a combination thereof. Incertain embodiments the bacterium may be one which expresses the cutC/Dgene cluster. The disclosure further provides a method of identifying acompound that inhibits TMA production. The method comprises contacting abacterium, such as a bacterium that is part of the gut microflora, or abacterial lysate that metabolizes choline to produce TMA with acandidate compound, such as a compound of Formula (I), and detecting TMA(or a metabolite thereof). In certain embodiments, the level of TMA (ormetabolite thereof) produced by the bacterium in contact with thecandidate compound is compared to (a) the level of TMA produced by abacterium or lysate not contacted with a candidate compound or known TMAinhibitor or (b) the level of TMA produced by the bacterium prior tocontact with the candidate compound. A reduction in the level of TMAproduced by the bacterium or lysate indicates that the candidatecompound inhibits conversion of choline to TMA.

A method of inhibiting the conversion of choline to TMA in vitro also iscontemplated. The method comprises contacting bacteria or bacteriallysate with one or more compounds of Formula (I). In variousembodiments, the bacteria comprise a single bacterial species or strain,or comprises a mixture of two or more (for example three, four, five, ormore) different bacterial species or bacterial strains. Similarly, abacterial lysate may be produced from a single bacterial species orstrain, or a mixture of two or more (for example three, four, five, ormore) different bacterial species or bacterial strains.

It will be appreciated that “inhibiting conversion of choline to TMA”does not require complete elimination of TMA production via cholinemetabolism. Any reduction in TMA formation from choline or a cholinerelated metabolite as a precursor is contemplated, e.g., at least 1%, atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% reduction;and also including from about 1% to about 100%, from about 10% to about90%, from about 20% to about 80%, from about 30% to about 70%, fromabout 40% to about 60%, and any combinations thereof.

In various embodiments, the inhibition of conversion of choline to TMAby the compounds of Formula (I) is not brought about by an antibioticmechanism of action, for example it is not brought about by anantibacterial mechanism of action, or by a mechanism of action whichreduces cell viability to 10% or lower, when compared to vehiclecontrol.

In certain embodiments of the invention, the amount of compound neededto provide 50% inhibition of conversion of choline to TMA is less thanthe amount of compound that reduces cell viability to 10% or lower, whencompared to vehicle control.

Any suitable method for measuring TMA in vitro or in vivo can be used inthe context of the invention. TMA, metabolites of TMA (including TMAO,DMA, or MMA), stable isotopes of TMA (such as deuterium labeled TMA,such as d3-, d6-, or d9-TMA), stable isotopes of TMAO (such as deuteriumlabeled TMAO, such as d3-, d6-, or d9-TMAO), stable isotopes of DMA(such as deuterium labeled DMA, such as d3-, or d6-DMA), stable isotopesof MMA (such as deuterium labeled MMA, such as d3-MMA), or choline(including stable isotopes of choline, for example d9-choline) can beassessed quantitatively or qualitatively. Exemplary methods of detectingand quantifying TMA are described in, for example U.S. Pub. No.2010/00285517, the disclosure of which is incorporated herein byreference in its entirety. For example, levels of TMA (or trimethylamineN-oxide (TMAO), DMA, or MMA) or choline are optionally measured via massspectrometry, ultraviolet spectroscopy, or nuclear magnetic resonancespectroscopy. Mass spectrometers include an ionizing source (such aselectrospray ionization), an analyzer to separate the ions formed in theionization source according to their mass-to-charge (m/z) ratios, and adetector for the charged ions. In tandem mass spectrometry, two or moreanalyzers are included. Such methods are standard in the art andinclude, for example, HPLC with on-line electrospray ionization (ESI)and tandem mass spectrometry.

In various embodiments, TMA or TMAO is measured in a biological samplefrom an individual. Biological samples include, but are not limited to,whole blood, plasma, serum, urine, feces, saliva, sweat, vaginal fluid,gingival crevicular fluid, or tissue. The sample may be collected usingany clinically-acceptable practice and, if desired, diluted in anappropriate buffer solution, heparinized, concentrated, or fractionated.Any of a number of aqueous buffer solutions at physiological pH, such asphosphate, Tris, or the like, can be used. Acidified buffers also may beused. For example, the final pH after adding buffer to sample mayoptionally be between pH 1 and pH 6, or between pH 1.5 and pH 3.0.

In addition, levels of TMA (or a metabolite or stable isotope thereof)or choline in the biological sample may be compared to a control value.The control value utilized will depend on the embodiment of theinvention. In certain embodiments, the control value may be the level ofTMA or TMAO produced in the individual (or by the bacterium) prior toadministration or exposure to a compound of Formula (I). In addition,the control value may be based on levels measured in comparable samplesobtained from a reference group, such as a group of individuals from thegeneral population, individuals diagnosed with a CVD or otherTMA-associated condition, individuals not previously diagnosed with aTMA-associated condition, nonsmokers, and the like, who have not beenexposed to a compound of Formula (I). Levels of TMA or TMAO or cholinemay be compared to a single control value or to a range of controlvalues. An individual is optionally identified as having an enhancedlevel of TMA prior to administration by comparing the amount of TMA in abiological sample from the individual with a control value.

The invention further provides a method of improving cardiovascularhealth of an individual. The method comprises administering to theindividual a composition comprising a compound set forth in Formula (I),as described above under the subheading “Compounds,” in an amounteffective to improve cardiovascular health. Cardiovascular health isassessed by testing arterial elasticity, blood pressure, ankle/brachialindex, electrocardiogram, ventricular ultrasound, platelet function (forexample platelet aggregation), and blood/urine tests to measure, forexample cholesterol, albumin excretion, C-reactive protein, or plasmaB-type peptide (BNP) concentration. In various aspects of the invention,administration of the compound of Formula (I) improves or maintains oneor more of the assay outcomes within normal ranges. Normal ranges ofoutcomes of each test are known in the art. Improvement incardiovascular health is, in some embodiments, marked by a reduction incirculating total cholesterol levels, reduction in circulating lowdensity lipoproteins (LDLs), reduction in circulating triglycerides, orreduction in blood pressure.

The invention also includes a method of improving a condition associatedwith conversion of choline to TMA in an individual in need thereof. Themethod comprises administering to an individual a composition comprisinga compound of Formula (I), in an amount effective to improve thecondition. “Improving a condition” refers to any reduction in theseverity or onset of symptoms associated with a disorder caused, atleast in part, by TMA. One of ordinary skill in the art will appreciatethat any degree of protection from, or amelioration of, a TMA-relateddisorder or symptom associated therewith is beneficial to an individual,such as a human. The quality of life of an individual is improved byreducing to any degree the severity of symptoms in an individual ordelaying the appearance of symptoms. Accordingly, a method in one aspectis performed as soon as possible after it has been determined that anindividual is at risk for developing a TMA-related disorder or as soonas possible after a TMA-related disorder is detected.

The condition associated with the conversion of choline totrimethylamine is, in various aspects of the invention, a cardiovasculardisease, trimethylaminuria, reduced or impaired kidney function, kidneydisease, chronic kidney disease, end-stage renal disease,trimethylaminuria, obesity or diabetes mellitus. The term“cardiovascular disease” (CVD) is used in the art in reference toconditions affecting the heart, heart valves, and vasculature (such asarteries and veins) of the body and encompasses diseases and conditionsincluding, but not limited to, arteriosclerosis, atherosclerosis,myocardial infarction, acute coronary syndrome, angina, congestive heartfailure, aortic aneurysm, aortic dissection, iliac or femoral aneurysm,pulmonary embolism, primary hypertension, atrial fibrillation, stroke,transient ischemic attack, systolic dysfunction, diastolic dysfunction,myocarditis, atrial tachycardia, ventricular fibrillation, endocarditis,arteriopathy, vasculitis, atherosclerotic plaque, vulnerable plaque,acute coronary syndrome, acute ischemic attack, sudden cardiac death,peripheral vascular disease, coronary artery disease (CAD), peripheralartery disease (PAD), cerebrovascular disease, adverse ventricularremodeling, ventricular systolic dysfunction, ventricular diastolicdysfunction, cardiac dysfunction, ventricular arrhythmia, and the like.

A condition may be atherosclerosis. Atherosclerosis involves theformation of atheromatous plaques that lead to narrowing (“stenosis”) ofthe vasculature, which can ultimately lead to partial or completeocclusion or rupture (aneurism) of the vessel, heart failure, aorticdissection, and ischemic events such as myocardial infarction andstroke. In various non-limiting embodiments, an inventive methodinhibits, reduces, or reverses (in whole or in part) the onset orprogression of atherosclerosis (for example reducing or preventinghardening or thickening of the arteries, plaque formation, endotheliumdamage, or arterial inflammation).

A condition may be trimethylaminurina. Trimethylaminuria (TMAU) is acondition characterized by an inability of individuals to convert TMA toTMAO, wherein affected individuals may have a fish-like body odorpresent in their urine, sweat or breath. (Yamazaki et al. Life Sciences(2004) 74: 2739-2747). Such individuals may benefit from a reduction inmetabolism of substrates including but not limited to choline, to TMA bybacteria in the gut. Individuals with TMAU or those wishing to reducetheir levels of TMA and TMAO, may also consume activated charcoal orcopper chlorophyllin, which act as sequestering agents, for example tomake TMA unavailable to transfer into the blood stream of an individual.Such sequestering agents may adsorb TMA, which is then excreted from thedigestive tract along with the sequestering agent.

The invention further provides the compounds of Formula (I) for use ininhibiting the conversion of choline to TMA in vivo or in vitro, forimproving or maintaining a condition associated with the conversion ofcholine to TMA; and use of the compounds of Formula (I) for inhibitingthe conversion of choline to TMA in vivo or in vitro, for improving ormaintaining a condition associated with the conversion of choline toTMA. As described previously, the present invention is based, at leastin part, on the discovery that compounds of Formula (I) inhibit cholinemetabolism by gut microbiota resulting in reduction in the formation ofTMA and trimethylamine N-oxide (TMAO). The disclosure providescompositions and methods that for example inhibit the conversion ofcholine to TMA in vitro and in vivo, improve or maintain cardiovascular,cerebrovascular, and peripherovascular health, and improve or prevent acondition associated with TMA and TMAO.

In various embodiments, administration of the compound of Formula (I)results in reduced TMA or TMAO levels, reduced total cholesterol levels,reduced LDL levels, increased HDL levels, reduced triglyceride levels,or normalized levels of other biomarkers associated with CVD (forexample excreted albumin, C-reactive protein, or plasma B-type peptide(BNP)). In some embodiments, the compound of Formula (I) reduces therisk of cardiovascular disease, trimethylaminuria, reduced or impairedkidney function, kidney disease, chronic kidney disease, end-stage renaldisease, trimethylaminuria, obesity, or diabetes mellitus, whenadministered to an individual.

Administration Regimens and Compositions

The amount of compound administered to the individual is sufficient toinhibit (in whole or in part) formation of TMA from choline. In variousaspects of the disclosure, the amount improves cardiovascular health orachieves a beneficial biological response with respect to an unwantedcondition associated with TMA (for instance the amount is sufficient toameliorate, slow the progression, or prevent a condition (such as CVD)).The effect can be detected by, for example, an improvement in clinicalcondition, reduction in symptoms, or by any of the assays or clinicaldiagnostic tests described herein. The precise effective amount for anindividual can depend upon the individual's body weight, size, andhealth; the nature and extent of the condition; and the compound orcombination of agents selected for administration. In various aspects,the amount of compound administered to an individual is about 0.001mg/kg to about 1000 mg/kg. Specific ranges of doses in mg/kg includeabout 0.1 mg/kg to about 500 mg/kg, about 0.5 mg/kg to about 200 mg/kg,about 1 mg/kg to about 100 mg/kg, about 2 mg/kg to about 50 mg/kg, andabout 5 mg/kg to about 30 mg/kg. An effective amount may be administeredto an individual as a single deployment of compound or as a divided dose(such as a single dose administered in multiple subunitscontemporaneously or close in time). An amount of compound may bedelivered one, two, or three times a day; one, two, or three times aweek; or one, two, three, or four times a month. The compound may bedelivered as a prodrug, which is converted to an active drug in vitro orin vivo.

A composition comprising the compound is administered by any route thatallows inhibition of choline conversion to TMA. A composition comprisingthe compound is, in various aspects of the invention, delivered to anindividual parenterally (for example intravenously, intraperitoneally,intrapulmonary, subcutaneously or intramuscularly), intrathecally,topically, transdermally, rectally, orally, sublingually, nasally or byinhalation. In various embodiments, a compound or a compositioncomprising a compound is administered to the gastrointestinal tract via,such as by ingestion. Sustained release formulations may also beemployed to achieve a controlled release of the compound when in contactwith body fluids in the gastrointestinal tract. Sustained releaseformulations are known in the art, and typically include a polymermatrix of a biological degradable polymer, a water-soluble polymer, or amixture of both, optionally with suitable surfactants.

The invention provides a composition comprising the compound of Formula(I) formulated with one or more physiologically acceptable excipients,carriers, stabilizers, tableting agents or diluents for use in themethods described herein. Excipients include, but are not limited to,carrier molecules that include large, slowly metabolized macromoleculessuch as proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, antioxidants (for exampleascorbic acid), chelating agents (for example EDTA), carbohydrates (forexample dextrin, hydroxyalkylcellulose, or hydroxyalkylmethylcellulose),liposomes, stearic acid, liquids (for example oils, water, saline,glycerol or ethanol), wetting or emulsifying agents, pH bufferingsubstances, and the like.

Compositions, such as for parenteral or oral administration, aretypically solids (for example, a lyophilized powder or cake), liquidsolutions, emulsions or suspensions, while inhalable compositions forpulmonary administration are generally liquids or powders. Exemplarydosage forms include, but are not limited to, tablets, troches,lozenges, aqueous or oil suspensions, non-aqueous solutions, powders,dispersible powders or granules (including micronized particles ornanoparticles), emulsions, hard or soft capsules, hard or softliquid-filled capsules, gelcaps, syrups, and elixirs. Solid dosecompositions, for example tablets or liquid filled capsules may beuncoated or may be coated by known techniques includingmicroencapsulation to delay disintegration and adsorption in thegastrointestinal tract. Solid dose compositions may be coated to targetdelivery to a specific region of the digestive tract. For example, thecomposition may be enteric coated to target delivery of the compositionto the small intestine, the large intestine, or to the colon. Additionalexemplary dosage forms may comprise coated microcapsules or coatedmicrobeads in a suspension or liquid chassis. In some embodiments, thecompound of Formula (I) is provided as a dietary (for example food ordrink) supplement. Dietary supplements are orally dosed and typicallycomprise vitamins, minerals, herbs or other botanicals, amino acids,enzymes, organ tissues, tissues from glands, or metabolites. Forexample, a compound of Formula (I) may be provided as a food in the formof a bar.

In some embodiments, the compounds described herein may be formulatedfor oral administration in a lipid-based composition suitable for lowsolubility compounds. Lipid-based compositions can generally enhance theoral bioavailability of such compounds. As such, the compositioncomprises in some aspects, an amount of a compound described hereintogether with at least one excipient selected from medium chain fattyacids and propylene glycol esters thereof (e.g., propylene glycol estersof edible fatty acids, such as caprylic and capric fatty acids) andphysiologically acceptable surfactants, such as polyoxyl 40 hydrogenatedcastor oil.

In some embodiments, the compounds described herein may be provided in adelayed release composition and are optionally released in a specificregion of the digestive tract of an individual. For example, thecomposition may be provided such that the compounds are released from anorally dosed composition in the distal portion of the digestive tractsuch as the ileum or the colon. In certain embodiments, the delayedrelease composition releases the compounds at a specific pH, or at arange of pH for targeted delivery within the digestive tract of anindividual. The compounds may be released, for example, between pH 6.0and pH 9.0, between pH 6.5 and pH 8.0, between pH 6.5 and pH 7.5,between pH 7.0 and pH 7.5, or between pH 7.0 and pH 8.0.

A method of the invention may comprise administering a second agent toan individual. The term “second agent” merely serves to distinguish theagent from the compound of Formula (I) and is not meant to limit thenumber of additional agents used in a method or denote an order ofadministration. One or more second agents are optionally incorporated inthe composition with the compound of Formula (I) administeredconcurrently but in separate dosage forms or administered separately intime.

Exemplary second agents include, but are not limited to, antimicrobials(such as antibiotics that kill bacteria in the gut); agents that improveintestinal motility (such as fiber or psyllium); agents that furtherreduce TMA levels in the gut including sequestering agents (such asactivated charcoal, or copper chlorophyllin); agents that further reduceTMA levels or production of TMA metabolites; agents that improve one ormore aspects of cardiovascular health, such as agents that normalizeblood pressure, decrease vascular inflammation, reduce plateletactivation, normalize lipid abnormalities; agents that promote theexcretion of TMA from the body; or agents that bind TMA so that itcannot be converted to TMAO. In various embodiments, the second agent isselected from the group consisting of Omega 3 oil, salicylic acid(aspirin), dimethylbutanol, garlic oil, garlic extract, olive oil, hilloil, Co enzyme Q-10, a probiotic, a prebiotic, a dietary fiber, psylliumhusk, bismuth salts, phytosterols, grape seed oil, green tea extract,vitamin D, an antioxidant (such as vitamin C and vitamin E), turmeric,curcumin, resveratrol, activated charcoal, or copper chlorophyllin. Incertain embodiments, the composition comprises dimethylbutanol orinhibitors of the formation of TMA from precursors other than choline(for example betaine, phosphatidylcholine, crotonobetaine, orcarnitine). Additional exemplary second agents are described in US2017/0151208, US 2017/0151250, US 2017/0152222, or US 2018/0000754,which are incorporated here by reference.

A method of the disclosure may further comprise administration of one ormore cardiovascular disease therapies. Examples of therapies include,but are not limited to, statins (e.g., Lipitor™ (atorvastatin),Pravachol™ (pravastatin), Zocor™ (simvastatin), Mevacor™ (lovastatin),and Lescol™ (fluvastatin)) or other agents that interfere with theactivity of HMGCoA reductase, nicotinic acid (niacin, which lowers LDLcholesterol levels), fibrates (which lower blood triglyceride levels andinclude, for example Bezafibrate (such as Bezalip®), Ciprofibrate (suchas Modalim®), Clofibrate, Gemfibrozil (such as Lopid®) and Fenofibrate(such as TriCor®)), bile acid resins (such as Cholestyramine, Colestipol(Colestid), and Cholsevelam (Welchol)), cholesterol absorptioninhibitors (such as Ezetimibe (Zetia®, Ezetrol®, Ezemibe®)),phytosterols such as sitosterol (Take Control (Lipton)), sitostanol(Benechol), or stigmastanol), alginates and pectins, lecithin, andnutraceuticals (such as extract of green tea and other extracts thatinclude polyphenols, particularly epigallocatechin gallate (EGCG),Cholest-Arrest™ (500 mg garlic and 200 mg lecithin). Cholestaway™ (700mg Calcium carbonate, 170 mg magnesium oxidem 50 μg chromiumpicolinate), Cholest-Off™ (900 mg of plant sterols/stanols), GuggulBolic (750 mg gugulipid (Commiphora mukul gum resin), and Kyolic® (600mg aged garlic extract and 380 mg lecithin)).

In related variations of the preceding embodiments, a compositioncomprising a compound of Formula (I) described herein, alone or incombination with one or more second agents(s), may optionally bearranged in a kit or package or unit dose, such as a kit or package orunit dose permitting co-administration of multiple agents. In anotheraspect, the composition comprising a compound of Formula (I) and the oneor more second agents are in admixture. In various embodiments, thecomponent(s) of the kit or package or unit dose are packaged withinstructions for administering the component(s) to an individual.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples, which are notintended to be limiting in any way.

Structures of representative compounds of Formula (I) are set forth inTABLE 1. Pharmaceutically acceptable counterions may include, but arenot limited to, chloride, bromide, or iodide, and salts and solvatesthereof.

TABLE 1 ID Structure SMILES Compound Name 1

C[N+](C)(CCF)CCl N-(chloromethyl)-2-fluoro-N,N- dimethylethan-1-aminium2

C[N+](C)(CCF)Cl 2-fluoro-N-(iodomethyl)-N,N- dimethylethan-1-aminium 3

C[N+](C)(CCF)CF (2-fluoroethyl)(fluoromethyl)dimethyl- l4-azane 4

C[N+](C)(CCF)CBr N-(bromomethyl)-2-fluoro-N,N- dimethylethan-1-aminium

EXAMPLES Example 1: Syntheses of Compounds

All synthesis procedures were performed at room temperature (RT) andatmospheric pressure unless stated otherwise.

The following are representative compounds of Formula (I):

Example 1.1: Synthesis ofN-(chloromethyl)-2-fluoro-N,N-dimethylethan-1-aminium chloride

In a 250 mL round bottomed flask equipped with a stirring bar, an oilbath and a reflux condenser, 10 g 2-Fluoroethylamine hydrochloride (CAS460-08-2) was added. 30 mL Formaldehyde solution (CAS 50-00-0, 37% inwater) was added dropwise by glass pipette over 10 minutes. 40 mL Formicacid (CAS 64-18-6) was added dropwise by glass pipette over 10 minutes.The reaction mixture was refluxed at 110° C. for 10 hours. After coolingthe reaction mixture to room temperature, 20 mL concentratedHydrochloric acid (CAS 7647-01-0, 36.5-38.0%) was added. After rotaryevaporation, the crude product (2-fluoro-N,N-dimethylethan-1-aminehydrochloric acid salt) was recrystallized in 2-propanol/diethyl ether.LC/MS: (ESI+) 92.

In a 250 mL round bottomed flask equipped with a stirring bar, 1 g2-fluoro-N,N-dimethylethan-1-amine hydrochloric acid salt, 0.6 g sodiumhydroxide (CAS 1310-73-2) and 40 mL ethanol/acetonitrile (l/l) wereadded. The reaction mixture was stirred at room temperature for 2 hours.The sodium chloride salt was removed using Chemrus disposable filterfunnel. To the filtrated solution, 1.5 g Silver tetrafluoroborate (CAS14104-20-2) and 2 mL Chloroiodomethane (CAS 593-71-5) were added. Thereaction mixture was stirred at room temperature for 24 hours. Themixture was passed through a pre-packed Amberlite IRA Cl-form column (50mL resin). The resin was further washed by methanol (100 mL). Afterrotary evaporation of the combined flow through, the oil-like productwas further washed by ether (3×100 mL) and then dried overnight on housevacuum (5-10 mm Hg) to get 1.5 g final product. LC/MS: (ESI+) 140.

Example 1.2: Synthesis of2-fluoro-N-(iodomethyl)-N,N-dimethylethan-1-aminium chloride

In a 250 mL round bottomed flask equipped with a stirring bar, an oilbath and a reflux condenser, 10 g 2-Fluoroethylamine hydrochloride (CAS460-08-2) was added. 30 mL Formaldehyde solution (CAS 50-00-0, 37% inwater) was added dropwise by glass pipette over 10 minutes. 40 mL Formicacid (CAS 64-18-6) was added dropwise by glass pipette over 10 minutes.The reaction mixture was refluxed at 110° C. for 10 hours. After coolingthe reaction mixture to room temperature, 20 mL concentratedHydrochloric acid (CAS 7647-01-0, 36.5-38.0%) was added. After rotaryevaporation, the crude product (2-fluoro-N,N-dimethylethan-1-aminehydrochloric acid salt) was recrystallized in 2-propanol/diethyl ether.LC/MS: (ESI+) 92.

In a 250 mL round bottomed flask equipped with a stirring bar, 1 g2-fluoro-N,N-dimethylethan-1-amine hydrochloric acid salt, 0.6 g sodiumhydroxide (CAS 1310-73-2) and 40 mL ethanol/acetonitrile (l/l) wereadded. The reaction mixture was stirred at room temperature for 2 hours.The sodium chloride salt was removed using Chemrus disposable filterfunnel. To the filtrated solution 1.5 g Silver tetrafluoroborate (CAS14104-20-2) and 0.76 mL Diiodomethane (CAS 75-11-6) were added. Thereaction mixture was stirred at room temperature for 24 hours. Themixture was passed through a pre-packed Amberlite IRA Cl-form column (50mL resin). The resin was further washed by methanol (100 mL). Afterrotary evaporation of the combined flow through, the crude product wasrecrystallized in 2-propanol/diethyl ether and then dried overnight onhouse vacuum (5-10 mm Hg) to get 1 g final product. LC/MS: (ESI+) 232.

Example 1.3: Synthesis of2-fluoro-N-(fluoromethyl)-N,N-dimethylethan-1-aminium chloride

In a 250 mL round bottomed flask equipped with a stirring bar, an oilbath and a reflux condenser, 10 g 2-Fluoroethylamine hydrochloride (CAS460-08-2) was added. 30 mL Formaldehyde solution (CAS 50-00-0, 37% inwater) was added dropwise by glass pipette over 10 minutes. 40 mL Formicacid (CAS 64-18-6) was added dropwise by glass pipette over 10 minutes.The reaction mixture was refluxed at 110° C. for 10 hours. After coolingthe reaction mixture to room temperature, 20 mL concentratedHydrochloric acid (CAS 7647-01-0, 36.5-38.0%) was added. After rotaryevaporation, the crude product (2-fluoro-N,N-dimethylethan-1-aminehydrochloric acid salt) was recrystallized in 2-propanol/diethyl ether.LC/MS: (ESI+) 92.

In a 250 mL round bottomed flask equipped with a stirring bar, 0.5 g2-fluoro-N,N-dimethylethan-1-amine hydrochloric acid salt, 0.2 g sodiumhydroxide (CAS 1310-73-2) and 40 mL ethanol/acetonitrile (l/l) wereadded. The reaction mixture was stirred at room temperature for 2 hours.The sodium chloride salt was removed using Chemrus disposable filterfunnel. To the filtrated solution 1 g Silver tetrafluoroborate (CAS14104-20-2) and 0.3 mL Fluoro(iodo)methane (CAS 373-53-5) were added.The reaction mixture was stirred at room temperature for 24 hours. Themixture was passed through a pre-packed Amberlite IRA Cl-form column (50mL resin). The resin was further washed by methanol (100 mL). Afterrotary evaporation of the combined flow through, the crude product wasrecrystallized in 2-propanol/diethyl ether and then dried overnight onhouse vacuum (5-10 mm Hg) to get 0.5 g final product. LC/MS: (ESI+) 124.

Example 2: Assay for Identifying and Characterizing Compounds thatInhibit the Formation of TMA from Choline

This example provides an exemplary assay for identifying andcharacterizing compounds that inhibit the formation of TMA from choline.

Proteus mirabilis 29906 (Pm) strain was grown aerobically overnight in500 ml of Nutrient Broth media (3 g/L beef extract, 5 g/L Peptone; Difco#234000) at 37° C. with 250 rpm shaking. The biomass was pelleted bycentrifugation at 6000×g for 12 minutes at 4° C. The cell pellet wassuspended in 240 mL of ice-cold 1× Phosphate Buffered Saline (Ca²⁺ andMg²⁺ free). Ninety micrograms of Lysozyme (Sigma# L6876 Lot# SLBG8654V;Sigma-Aldrich Corp., St. Louis, Mo.) was added and incubated with 320rpm shaking for 30 minutes at 4° C. Lysis was achieved via French presswith a 4° C. prechilled 1″ diameter chamber at 1000 psi (high ratio;internal PSI equivalent ˜16000). The lysate was centrifuged at 6,000×gfor 12 minutes at 4° C. to pellet extra debris. A protein concentrationof the centrifuged lysate supernatant was determined by a BCA ProteinAssay Kit (Pierce #23225; Thermo Fisher Scientific Co., Waltham, Mass.)and protein concentration adjusted to 3 mg/ml with 1×Dulbecco'sphosphate buffered saline (DPBS). The centrifuged supernatant lysate wasaliquoted into 20 mL volumes and stored frozen at −80° C.

Proteus mirabilis 29906 (Pm) lysate was diluted to 1.5 mg/mL proteinwith 1×DPBS. Choline chloride (CC) (1M stock) was added to reach a finalconcentration of 2.5 mM choline chloride. The mixture was mixed using avortex mixer for approximately 15 seconds and incubated at 37° C. for 22hours. After incubation, 150 μL of CC-treated Pm lysate was dispensedinto a deep-well plate (polypropylene, 2 mL volume, Corning Axygencatalogue # P-DW-20-C). Candidate IC₅₀ compounds from TABLE 1 andvehicle control (respective vehicle control of DMSO or water), orcontrol compounds (IC50 control, 8-Quinolinol hemisulfate salt (SigmaCatalog #55100)) were added at a 1:100 dilution (e.g., 1.5 μL per well).The plates were agitated on a plate shaker for 1 minute. d9-cholinechloride (1.5 μL of 5 mM) was added to all wells to reach a finald9-choline chloride concentration of 50 μM.

The plates were again agitated on a plate shaker for 1 minute andincubated at 37° C. for two hours. After incubation, 1.5 μL of formicacid was added to each well (final concentration=1% formic acid). Theplates were agitated on a plate shaker for 1 minute and placed on ice.Cell lysate samples were spiked with stable isotope labeled internalstandard (22.5 μL of 6 μg/mL of 13C3-trimethylamine (13C3-TMA) was addedto each sample), then d9-trimethylamine (d9-TMA), trimethylamine (TMA)and 13C3-TMA were isolated from the lysate after protein precipitationas described below. Acetonitrile acidified with 0.1% formic acid, 600μL, was added to each sample which was then centrifuged (2,100 g for 20minutes) to pellet the protein and other precipitates. The supernatantwas removed and analyzed as described below. The TMA, d9-TMA and13C3-TMA in the isolated supernatant samples were subjected to gradientHigh Performance Liquid Chromatography (HPLC) analysis on a WatersAtlantis HILIC Silica column, from Waters Corp., Milford, Mass., (2.1×50mm, 3 μm particles) with an Atlantis Silica HILIC Sentry guard column,from Waters Corp., Milford, Mass., (100 Å, 3 μm, 2.1 mm×10 mm), 10 mMammonium formate in water with 0.1% formic acid as mobile phase A and0.1% formic acid in acetonitrile as mobile phase B. Detection andquantitation was achieved by tandem mass spectrometry operating undermultiple reaction monitoring (MRM) MS/MS conditions (m/z 60.1→44.1 forTMA, m/z 69.1→49.1 for d9-TMA, m/z 63.0→46.1 for 13C3-TMA). TMA andd9-TMA calibration standards (STD), prepared in 80/20/0.1%acetonitrile/Water/Formic Acid, were used to construct a regressioncurve by plotting the response (peak area TMA/peak area 13C3-TMA) versusconcentration for each standard. The concentrations of TMA and d9-TMA inthe cell lysate were determined by interpolation from the quadratic(1/×2) regression curve.

Example 2 Provides Exemplary Methods of Identifying and Quantitating TMAin A Sample, as Well as Screening Candidate Inhibitory Compounds

IC₅₀ measurements for inhibition of conversion of choline to TMA, asoutlined in EXAMPLE 2, for representative compounds of Formula (I), areset forth in TABLE 2.

TABLE 2 TMA Inhibition (IC₅₀, ID Name SMILES mol/L) 1N-(chloromethyl)-2-fluoro- C[N+](C)(CCF)CCl•[Cl] 2.700E−07N,N-dimethylethan-1- aminium chloride 2 2-fluoro-N-(iodomethyl)-C[N+](C)(CCF)CI•[Cl] 1.786E−07 N,N-dimethylethan-1- aminium chloride 3(2-fluoroethyl) C[N+](C)(CCF)CF•[Cl] 2.113E−07 (fluoromethyedimethyl-14-azane chloride

Example 3 Polymicrobial Screening Method

Human fecal polymicrobial incubation with deuterium labeled cholinecompound screening method, including cell viability assay. All materialswere pre-reduced in an anaerobic chamber for 24 hours before using inthe experiments and experimental procedures were performed underanaerobic conditions (chamber purged with 85% nitrogen, 5% hydrogen, 10%carbon dioxide).

Human fecal samples were collected from a healthy male volunteer with nochronic illnesses, blood borne diseases or active infections. Thevolunteer had not received antibiotics within two months prior todonation and provided written informed consent. Samples were diluted tomake a 20% (w/v) fecal slurry by resuspension of the feces in a mediacontaining 3% (w/v) tryptic soy broth, 1% (w/v) trehalose, pH 7.3. Thefecal slurry was homogenized and filtered by hand using a stomacher bagwith an integrated 170 μm membrane. DMSO (5% (w/v)) was added to thefiltered slurry and aliquots were stored in cryogenic vials at −80° C.until use. Frozen fecal slurries were diluted to 0.2% (w/v) with M9media (Na₂HPO₄ (6 g/L), KH₂PO₄ (3 g/L), NaCl (0.5 g/L) with addition of0.1 mM CaCl₂) and 1 mM MgSO₄) and dispensed (1 mL) into deep well96-well plates. Diluted fecal slurries containing 50 μM d9-cholinechloride and compounds in doses ranging from 500 μM to 3.81 nM weresealed and incubated at 37° C. with shaking. After 20 hours, an aliquotof the fecal polymicrobial community was analyzed for viability usingPrestoBlue cell viability reagent (Thermo Fisher Scientific, USA) asdescribed below. The reaction plates were subsequently centrifuged(4000× g at 4° C. for 12 min) to pellet fecal material and 150 μlaliquots were transferred and quenched with addition of formic acid to1% (v/v). All fecal processing and polymicrobial assay steps wereperformed in an anaerobic environment. The products were determined byLC/MS/MS and IC₅₀ values were calculated as described previously fordetection and analysis of TMA and d9-TMA in EXAMPLE 2. Inhibition ofconversion of choline to TMA is calculated as IC₅₀ (mol/Liter)

For the PrestoBlue cell viability assay, a 6 μL aliquot of the fecalpolymicrobial community assay was added to 84 μL M9 media in a black,clear bottom 96 well plate. To this was added 10 μL of PrestoBluereagent, covered and shaken for 1 minute at 800 rpm. The plates wereincubated at 37° C. for 30 minutes and fluorescence read following themanufacturer's instructions. Cell viability was calculated as %fluorescence compared to vehicle control (e.g. 1% DMSO).

EXAMPLE 3 provides exemplary methods of screening candidate inhibitorycompounds for the conversion of choline to TMA and for calculation ofcell viability.

Example 4 Preclinical Screening Method

Starting at day 0, mice (C57bl/6, ˜19 g, 10 wk of age; n=5/group) weremaintained in accordance with the NIH guidelines in a 12:12 hrlight:dark cycle and provided with 1% Choline Added diet (Envigo customformulation prepared, similar to Teklad Global Rodent Diet 2018) adlibitum. Concurrent with introduction of the diet, mice were gavagedonce daily orally using a 1.5″ 22 G ball-tip curved feeding needle toadminister compound in 200 μl or less of water at one or multiple of thedose 0, 1.0, 3.1, 10, 31, 100 or 310 mg/kg/day. Urine was collected oncedaily in the morning. Animals were restrained by hand and bladder wasexpressed by gentle palpation of the pelvic region. Aliquots of 1-5 μlof urine were centrifuged at 1,300×g for 5 min in a 1.5 mL conicalbottom tube with a snap top, to precipitate any potential cellulardebris, and supernatants were transferred to a clean screw-cap tube witho-ring seal and stored at −80° C. until analysis. Sixty microliters orless of blood was collected at 20 hours post gavage, into a heparinizedcapillary tube. Blood was kept at 4° C., then spun using a centrifuge (5min in centrifuge designed to capillary tubes) to separate plasma andhematocrit within 4 hours after collection. Plasma samples were storedat −80° C.

Measurements of Choline Metabolites:

For measurement of TMA in plasma, samples were acidified (10 mM HClfinal) prior to storage at −80° C. TMAO and trimethylamine (TMA) andtheir d9-isotopologues were quantified using stable isotope dilutionHPLC with on-line electrospray ionization tandem mass spectrometry(LC/EST/MS/MS) methods as described in (Wang Z, Klipfell E, Bennett B J,et al. (2011) Gut flora metabolism of phosphatidylcholine promotescardiovascular disease. Nature 472:57-63) using d4(1,1,2,2)-choline,d3(methyl)-TMAO, and d3(methyl)-TMA as internal standards.Concentrations of TMAO in urine were adjusted for urinary dilution byanalysis of urine creatinine concentration. Samples were taken atdifferent days during the studies and different doses were administeredto avoid side effects at higher doses of some of the compounds.

EXAMPLE 4 provides exemplary methods of screening candidate inhibitorycompounds for the conversion of choline to TMA. Inhibition is calculatedas remaining plasma TMAO as a percentage of plasma TMAO in same dayvehicle control.

Example 5: Additional In Vitro Assay for Identifying and CharacterizingCompounds that Inhibit the Formation of TMA from Choline

Ability of compounds to inhibit the conversion of choline to TMA in celllysates or whole cells were determined using methods as described inWang, Z, Roberts, A B, Buffa J A, et al. (2015) Non-lethal inhibition ofgut microbial trimethylamine production for the treatment ofatherosclerosis, Cell 163: 1585-1595. Briefly, efficacy was measured asIC50 (nM) by inhibition of conversion of choline to TMA metabolized byrecombinant P. mirabilis Cut C/D lysate; recombinant D. alaskensis CutC/D lysate, or whole cell wild-type P. mirabilis.

IC₅₀ measurements for inhibition of conversion of choline to TMA, asoutlined in EXAMPLE 5, for representative compounds of Formula (I) areset forth in TABLE 3.

TABLE 3 IC50 (M) IC50 (M) EC50 (M) recombinant recombinant whole P.mirabilis D. alaskensis cell wild-type ID Compound Name Cut C/D lysateCut C/D lysate P. mirabilis 1 N-(chloromethyl)-2-fluoro-N,N- 6.90E−077.60E−07 8.70E−06 dimethylethan-1-aminium chloride 22-fluoro-N-(iodomethyl)-N,N- 2.40E−07 1.50E−07 6.70E−05dimethylethan-1-aminium chloride 3(2-fluoroethyl)(fluoromethyl)dimethyl- 8.70E08 1.70E−07 7.20E−0614-azane chloride

EXAMPLE 5 provides exemplary methods of identifying and quantitating TMAin a sample, as well as screening candidate inhibitory compounds. Allcompounds in TABLE 3 were found to inhibit the conversion of choline toTMA.

Example 6: Rapid Preclinical Method to Determine Compound Efficacy

Challenge: C57bl/6 female mice (8 wk of age ˜20 g BW) were maintained inaccordance with the NIH guidelines in a 12:12 hr light:dark cycle onnormal chow diet were placed in a clean cage without food ˜1 hr prior togavage. Mice were given 2 mg d9-Choline+x mg/kg inhibitor (where x=0 to310 mg/kg) in water by oral gavage using a 1.5″ 22 G ball-tip curvedfeeding needle to administer compound in 200 μl of water. Food wasreturned after a 2 hr fast (1 hr after gavage administration). Blood (30μL) was collected into a heparinized capillary tube at 2, 3 and 4 hoursafter gavage. Blood was kept at 4° C., then spun using a centrifuge (5min in centrifuge designed to capillary tubes) to separate plasma andhematocrit within 4 hours after collection. Plasma samples were storedat −80° C. Concentration of d9 Choline, d9TMA and d9TMAO was measured byLC-MS/MS.

Flora Normalization: Twenty four hours post-gavage mice were placed in aclean cage and fecal material from conventional mice was spread in allthe cages.

EXAMPLE 6 provides exemplary methods of screening candidate inhibitorycompounds for the conversion of choline to TMA.

EC₅₀ measurements for inhibition of conversion of choline to TMA, asoutlined in EXAMPLE 6, for representative compounds of Formula (I) areset forth in TABLE 4.

TABLE 4 Calculated EC₅₀ (mg/kg) compared to Vehicle Control, asdescribed in EXAMPLE 6 EC₅₀ ID Compound Name (mg/kg) Time 1N-(chloromethyl)-2-fluoro-N,N-dimethylethan- 3.10 3 hr 1-aminiumchloride 2 2-fluoro-N-(iodomethyl)-N,N-dimethylethan- 15.40 3 hr1-aminium chloride 3 (2-fluoroethyl)(fluoromethyedimethyl-14-azane 1.103 hr chloride

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application and any patent application or patent to which thisapplication claims priority or benefit thereof, is hereby incorporatedherein by reference in its entirety unless expressly excluded orotherwise limited. The citation of any document is not an admission thatit is prior art with respect to any invention disclosed or claimedherein or that it alone, or in any combination with any other referenceor references, teaches, suggests or discloses any such invention.Further, to the extent that any meaning or definition of a term in thisdocument conflicts with any meaning or definition of the same term in adocument incorporated by reference, the meaning or definition assignedto that term in this document shall govern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A composition comprising a physiologicallyacceptable carrier and a compound of Formula (I):

wherein: W is selected from F, Cl, Br or I; X⁻ is a pharmaceuticallyacceptable anion, and including any acceptable salts or solvatesthereof.
 2. The composition of claim 1 wherein the compound is selectedfrom the group consisting of:N-(chloromethyl)-2-fluoro-N,N-dimethylethan-1-aminium chloride,2-fluoro-N-(iodomethyl)-N,N-dimethylethan-1-aminium chloride,(2-fluoroethyl)(fluoromethyl)dimethyl-14-azane chloride andN-(bromomethyl)-2-fluoro-N,N-dimethylethan-1-aminium chloride, andincluding any acceptable salts or solvates thereof.
 3. The compositionof claim 1 wherein W is F, X⁻ is selected from Cl⁻, Br⁻ or I⁻, includingany acceptable salts or solvates thereof.
 4. A method of inhibiting theconversion of choline to trimethylamine (TMA) and reducing TMAO level inan individual comprising administering to the individual a compositioncomprising a compound set forth in Formula (I):

wherein: W is selected from F, Cl, Br or I; X⁻ is a pharmaceuticallyacceptable anion, and including any acceptable salts or solvatesthereof; wherein the compound is administered in an amount effective toinhibit formation of trimethylamine (TMA) from choline in theindividual.
 5. The method of claim 4 wherein W is F, X⁻ is selected fromCl⁻, Br⁻ or I⁻, and including any acceptable salts or solvates thereof.6. The method of claim 4 wherein the compound is selected from the groupconsisting of: N-(chloromethyl)-2-fluoro-N,N-dimethylethan-1-aminiumchloride, 2-fluoro-N-(iodomethyl)-N,N-dimethylethan-1-aminium chloride,(2-fluoroethyl)(fluoromethyl)dimethyl-14-azane chloride andN-(bromomethyl)-2-fluoro-N,N-dimethylethan-1-aminium chloride, andincluding any acceptable salts or solvates thereof.
 7. The method ofclaim 4 comprising administering to the individual a second agentselected from the group consisting of Omega 3 oil, salicylic acid,dimethylbutanol, garlic oil, olive oil, krill oil, Co enzyme Q-10, aprobiotic, a prebiotic, dietary fiber, psyllium husk, bismuth salts,phytosterols, grape seed oil, green tea extract, vitamin D, anantioxidant, turmeric, curcumin, and resveratrol.
 8. The method of claim4, comprising administering the compound to an individual having anelevated level of TMAO in blood, plasma, serum, or urine, andcombinations thereof.
 9. A method of improving a condition associatedwith the conversion of choline to trimethylamine (TMA) in an individualcomprising administering to the individual a compound set forth inFormula (I):

wherein: W is selected from F, Cl, Br or I; X⁻ is a pharmaceuticallyacceptable anion, and including any acceptable salts or solvatesthereof; wherein the compound is administered in an amount effective totreat or prevent the disease or condition associated withcholine-related trimethylamine (TMA) in the individual.
 10. The methodof claim 9, wherein the condition associated with choline-relatedtrimethylamine is a cardiovascular disease, trimethylaminuria, reducedor impaired kidney function, kidney disease, chronic kidney disease,end-stage renal disease, or diabetes mellitus.
 11. The method of claim10, wherein the cardiovascular disease is selected from the groupconsisting of angina, arrhythmia, atherosclerosis, cardiomyopathy,congestive heart failure, coronary artery disease (CAD), carotid arterydisease, endocarditis, coronary thrombosis, myocardial infarction (MI),high blood pressure/hypertension, hypercholesterolemia/hyperlipidemia,mitral valve prolapsed, peripheral artery disease (PAD), and stroke.